This project conducts clinical trials of live intranasal pediatric vaccine candidates for human respiratory syncytial virus (RSV), human parainfluenza virus serotypes 1, 2, and 3 (HPIV1, 2, and 3), and human metapneumovirus (HMPV), with RSV being the most important vaccine target. RSV vaccine: The primary goal is to identify, in phase 1 pediatric clinical trials, one or more live-attenuated strains of RSV suitable for further development as a pediatric RSV vaccine, to be given by intranasal administration. We previously showed that deletion of the ORF encoding the small (90 amino acids) viral M2-2 protein (delM2-2) results in down-regulated viral RNA replication (causing viral attenuation) and up-regulated viral gene transcription and antigen synthesis, raising the possibility of increased immunogenicity. We have been evaluating several versions of delM2-2 viruses phase 1 clinical trials in RSV-seronegative children 6-24 months of age. First, a prototype virus called RSV MEDI/delM2-2 was evaluated with clinical collaborators at Johns Hopkins University School of Public Health (JHU; Clinicaltrials.gov identifier NCT01459198). This showed that this virus was very highly attenuated for replication, and possibly was more attenuated than necessary. Nonetheless, compared to our previous lead RSV vaccine candidate, called rA2cp248/404/1030delSH, the MEDI/delM2-2 virus induced significantly higher titers of RSV-neutralizing serum antibodies. Thus, it indeed appeared to have increased immunogenicity per infectious unit. Surveillance during the subsequent RSV season provided presumptive evidence of protection and strong anamnestic antibody responses. A second delM2-2-based candidate, called RSV LID/M2-2, was evaluated in a similar phase 1 study by JHU plus the International Maternal Pediatric Adolescent AIDS Clinical Trials (IMPAACT) network (NCT02040831 and NCT02237209). Unexpectedly, the LID/delM2-2 virus was substantially less restricted than MEDI/delM2-2 and was deemed to be insufficiently attenuated. The basis for the difference in replication by these two viruses is presently being investigated. The LID/delM2-2 virus was more immunogenic than MEDI/delM2-2, presumably due to the increased replication. There was no evidence of increased respiratory tract illness in vaccine versus placebo recipients except that one vaccinee had brief, mild LRT illness. This was concurrent with the shedding of vaccine virus as well as enterovirus/rhinovirus. Therefore, causality was unclear. Surveillance during the subsequent RSV season provided presumptive evidence of protection and strong anamnestic antibody responses. Several other versions of RSV delM2-2 viruses presently are under evaluation in similar phase 1 trials, namely RSV D46/cp/delM2-2 (NCT02601612), RSV LID/delM2-2/1030s (NCT02794870 and NCT0252339), RSV LID/cp/delM2-2 (NCT02890381 and NCT02948127), and RSV D46/NS2/N/delM2-2 (NCT03099291 and NCT03102034). Ideally, we would like to identify a delM2-2 mutant with replication properties intermediate between the MEDI/delM2-2 and LID/delM2-2 viruses, with the idea that it would be desirable to have as much replication as is safe in order to enhance immunogenicity. The rA2cp248/404/1030delSH virus mentioned above, which was a previous lead candidate, contains a series of point mutations and deletion of the SH gene, and is highly temperature-sensitive. However, in a previous phase 1 clinical trial in seronegative infants and children 6-24 months of age, it exhibited genetic instability that primarily involved two attenuating point mutations in the L protein called 248 and 1030. We previously developed new versions of these two mutations that were modified for increased genetic stability (called 248s and 1030s). A version of the rA2cp248/404/1030delSH virus was created that incorporated these stabilized mutations, resulting in a virus called RSV cps2. This virus has now been evaluated in a phase 1 pediatric clinical trial in seronegative children 6-24 months of age (NCT01852266 and NCT01968083), in collaboration with JHU and IMPAACT. Consistent with the results from its parent, this virus was highly attenuated but was less immunogenic than the delM2-2-based viruses noted above. Sequence analysis of shed vaccine virus from vaccinees indicated an absence of de-attenuation at the stabilized attenuating mutations 248s or 1030s, indicating that substantial stabilization of these mutations indeed had been achieved. Thus, the cps-2 virus is a feasible vaccine candidate, although at the present time we are focusing on viruses like the delM2-2 viruses that may be more effective. The stabilized 1030 mutation presently is being used in other vaccine candidates (such as LID/delM2-2/1030s noted above). We are evaluating another RSV vaccine candidate called RSV delNS2/del1313/I1314L. This virus contains the deletion of the nonstructural protein 2 (NS2) gene, whose encoded protein antagonizes host responses to viral infection, notably the type I interferon (IFN) response. It also contains a stabilized mutation in the polymerase L protein that we previously developed, and which consists of deletion of codon 1313 combined with a I1314L missense mutation. This virus is being evaluated in a phase 1 pediatric clinical trial (NCT01893554) presently in progress. Thus, we have several promising RSV candidates in phase 1 pediatric clinical trials. Our goal is to identify two lead candidates to bring forward to larger studies in 2018. Wild type (wt) RSV: We prepared a clinical trial lot of wt RSV strain A2 produced from cDNA. This provides a virus with a well-defined passage history and reduced possibility of adventitious agents. In collaboration with the Medical Virology Section, this virus presently is being evaluated in an in-patient setting for infectivity, replication, pathogenesis, and immunogenicity in healthy adult volunteers in a dose-escalation study (NCT02484417). This will provide an infection model that can be used to evaluate anti-RSV drug candidates and adult RSV vaccine candidates, and to study viral pathogenesis and the host response. HPIV1 vaccine: We previously developed and evaluated an HPIV1 vaccine candidate called rHPIV1-C(R84G/del170)HN(T553A)L(Y942A), which includes stabilized mutations, in seronegative children 6 to 59 months of age (NCT00641017) and showed that it is over-attenuated. Over-attenuation can be corrected by reverse genetics. HPIV2 vaccine: We developed and are evaluating an HPIV2 vaccine candidate called rHPIV2-V94(15C)/948L/1724, which includes stabilized mutations, in seronegative children 6 to 59 months of age (NCT01139437). HMPV vaccine: We previously developed an HMPV vaccine virus called rHMPV-Pa in which the HMPV P gene was replaced by that of avian MPV, thus conferring a host range attenuation phenotype. A phase 1 trial in seronegative children 6-59 months of age (NCT01255410) showed that this virus is over-attenuated. This provides a benchmark for pre-clinical comparison with other existing vaccine candidates to identify ones that are less attenuated. These were the first live-attenuated HPIV1, 2, and HMPV vaccines to be evaluated in humans.